Thymidylate synthetase is the target of certain clinically useful cancer chemotherapeutic agents. We intend to elucidate in detail the mechanism of this enzyme, as well as the mechanism of its interaction with 5-fluoro-2'-deoxyuridylate. Unusual interactions of this enzyme with 5-trifluoro- and 5-formyl-deoxyuridylate and 1-(beta-D-arabinosyl)5-fluorouracil shall also be investigated, and new inhibitors of this enzyme shall be designed based on what is known of its mechanism. We shall also purify mammalian thymidylate synthetases by affinity-chromatography and compare their properties to those of the much studied enzyme from L. casei. We have formulated a hypothesis that the mechanism of thymidylate synthetase may serve as a paradigm for many enzymes involved in pyrimidine metabolism. We believe that nucleophilic attack at the 6-position of the pyrimidine heterocycle may be a general feature of many enzymes which modify the 4- and 5-positions of pyrimidine heterocycles and cleave the glycosidic bond of pyrimidine nucleosides/nucleotides. This hypothesis shall be tested using methodology resulting from studies of thymidylate synthetase. Should a fraction of these enzymic reactions proceed by the proposed mechanism, a chemically and teleologically congruent mechanism would be established for a large number of diverse reactions of pyrimidine metabolism.